The present invention relates generally to toxin binders for animal feed applications and, more specifically, to the application of a novel microporous zeolite as a multi toxin binder in animal feeds.
Mycotoxins are invisible, odorless and cannot be detected by smell or taste, but can result in great economic losses at all levels of agricultural feed production and especially in animal production. Mycotoxins are secondary metabolites produced by filamentous fungi such as Fusarium, Aspergillus, and Penicillium prior to and during harvest, or during (improper) storage. Their toxic effects are very diverse depending upon the mycotoxins (Akande, K. E., Abubakar, M. M., Adegbola, T. A., and Bogoro, S. E. 2006. Nutritional and Health Implications of Mycotoxins in Animal Feeds: A Review. Pakistan Journal of Nutrition, 5: 398-403). In farm animals, mycotoxins have negative effects on feed intake, animal performance, reproductive rate, growth efficiency, and immunological defense as well as been carcinogenic, mutagenic, teratogenic, cause tremors or damage the central nervous system, hemorrhagic, as well as causing damage to the liver and kidneys. Mycotoxins are metabolized in the liver and the kidneys and also by microorganisms in the digestive tract. Therefore, often the chemical structure and associated toxicity of mycotoxin residues excreted by animals or found in their tissues are different from the parent molecule (Ratcliff, J. Aug. 16, 2002. The Role of Mycotoxins in Food and Feed Safety. Presented at Animal Feed Manufacturers Association). Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions (Sohn, H. B., Seo, J. A., and Lee, Y. W. 1999. Co-occurrence of Fusarium Mycotoxins in Mouldy and Healthy Corn from Korea. Food Additives and Contaminants. 16: 153-158). Considering this synergic effect, it is very likely, that animals are exposed to mixtures rather than to individual compounds. Field studies have shown that more severe toxicosis in animals can result from the additive and synergistic effects of different mycotoxins (Ratcliff J., 2002. The role of mycotoxins in food and feed safety. Presented at AFMA (Animal Feed Manufacturers Association) on 16 Aug. 2002). The problem of mycotoxins does not just end in animal feed or reduced animal performance; many become concentrated in meat, eggs and milk of animal and can pose a threat to human health. There is an increasing concern about levels of mycotoxins in human foods, both from vegetable origin and animal origin.
Although there are geographic and climatic differences in the production and occurrence of mycotoxins, exposure to these substances is worldwide. Mycotoxins are estimated to affect as much as 25 percent of the world's crops every year (Akande K. E., Abubakar M. M., Adegbola T. A. and Bogoro S. E. 2006. Nutritional and Health Implications of Mycotoxins in Animal Feeds: A Review. Pakistan Journal of Nutrition. 5 (5): 398-403). Most countries have stringent regulations on mycotoxin levels in feed and the main goal of agricultural and food industries are the prevention of mycotoxin contamination in the field. Management practices to maximize plant performance and decrease plant stress can decrease mycotoxin contamination substantially. This includes planting adapted varieties, proper fertilization, weed control, necessary irrigation, and proper crop rotation (Edwards, S. G. 2004. Influence of Agricultural Practices on Fusarium Infection of Cereals and Subsequent Contamination of Grain by Tricothecenes Mycotoxins. Toxicology Letters, 153: 29-35). But even the best management strategies cannot eliminate mycotoxin contamination in years favorable for disease development. Among the various mycotoxins identified especially affecting the agricultural and food industries, some occur significantly in naturally contaminated foods and feeds. It includes aflatoxin B1 (afla B1), ochratoxin A (OTA), zearalenone (zea), mycophenolic acid (MPA), cyclopiazonic acid (CPA), fumonisin B1 (fum B1), tricothecenes (T-2), deoxynivalenol (DON) and patulin (pat).
Afla B1, a metabolite of fungus Aspergillus flavus and Aspergillus parasiticus, is an extremely hepatotoxic compound that frequently contaminates poultry feeds at low levels (Ramos A. J., Hernandez E. 1996. In vitro aflatoxin adsorption by means of a montmorillonite silicate. A study of adsorption isotherm. Animal Feed Technology. 62: 263-269).
Another family of mycotoxins produced by Penicillium and Aspergillus genera is OTA, being the most potent toxin, adversely affects production parameters and the health of poultry. This mycotoxin is known to be a nephrotoxic, immunotoxic, carcinogenic, and teratogenic substance to a variety of animal species. Intestinal injuries including inflammation and diarrhea were seen on ingestion of OTA (Maresca M., Mahfoud R., Pfohl-Leszkowicz A and Fantini J. 2001. The mycotoxin ochratoxin A alters intestinal barrier and absorption functions but has no effect on chloride secretion. Toxicology and Applied Pharmacology. 176: 54-63).
Fusarium species which produces mycoestrogen zea, reported to activate estrogen receptors which results in functional alteration in reproductive organs. Swelling of the vent and increase in oviduct size could be asssociated with high levels of zea (Fink-Gremmels J, Malekinej ad H. 2007. Clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone. Animal Feed Science and Technology. 137:326-341.)
Penicillium roqueforti is one of the most important sources of MPA and it occurs predominantly in maize (Mansfield M. A., Jones A. D. and Kuldau G. A. 2008. Contamination of fresh and ensiled maize by multiple Penicillium mycotoxins. Department of Plant Pathology, the Pennsylvania State University, University Park 16802, USA. Phytopathology. 98: 330-6) and silage (Schneweis I., Meyer K., Hörmansdorfer S. and Bauer J. 2000. Mycophenolic Acid in Silage. Appl Environ Microbiol. 66: 3639-3641). Penicillium roqueforti, Penicillium rubrum, and Penicillium brevicompactum are associated with MPA production and found in both cattle and poultry feeds (Koteswara Rao V., Shilpa P., Girisham S and Reddy S. M. 2011. Incidence of mycotoxigenic Penicillia in feeds of Andhra Pradesh, India. International Journal for Biotechnology and Molecular Biology Research. 2: 46-50). The mycotoxin MPA has strong immunosuppressive action, which occurs by blocking the conversion of Inosine-5-phosphate to guanosine-5-phosphate (Allison A. C. and Eugui E. M. 2000. Mycophenolate mofetil and its Mechanism of action. Immunopharmacology. 47: 85-118).
The mycotoxin CPA is also produced by fungi belonging to the genus Aspergillus and Penicillium. The co-occurrence of CPA with afla B1 is mainly due to the growth of Aspergillus flavus which produces both these toxins (Dilek H., Sukra S., Funda K H and Nesirin M. 2012. Natural contamination of Cyclopiazonic acid in dried figs and co-occurrence of aflatoxin. Food control. 23: 82-86). The CPA toxicity in poultry also causes pathological effects such as hyperemia and ulceration of the proventriculus, focal necrosis in the liver and spleen, lymphoid depletion of the bursa of fabricius, weight loss and changes in relative organ weight (Gentles A., Smith E. E., Kubena L. F., Duffus E., Johnson Paul., Thompson J., Harvey R. B and Edrington T. S. 1999. Toxicological evaluations of Cyclopiazonic acid and Ochratoxin A in broilers. Poultry science. 78: 1380-1384).
Fum B1, a toxic compound was reported to be produced by Fusarium moniliforme (Gelderblom, W. C. A., Jeskiewicz, K., Marasas, W. F. O., Thiel, P. G., Horak, R. M. m Vleggaar, R., and Kriek, N. P. J. 1988. Fumonisins-novel, mycotoxins with cancer promoting activity produced by Fusarium moniliforme. Appl. Environ. Microbiol. 54: 1806-1811). The effects of this toxin includes rickets and immunosuppression in poultry (Norred, W. P. 1993. Fumonisins mycotoxins produced by Fusarium moniliforme. J. Toxicol. Environ. Health. 38:309-328). However the ill effects of this toxin were noted in other animal species also. Fum B1 was also reported to co-occur with afla B1 in Indian maize and poultry feeds (Prathapkumar H. Shetty and Ramesh V. Bhat. 1997. Natural Occurrence of Fumonisin B1 and Its Co-occurrence with Aflatoxin B1 in Indian Sorghum, Maize, and Poultry Feeds. J. Agric. Food Chem. 45: 2170-2173).
T-2 toxin produced by Fusarium fungi exerts toxic effects in poultry species also. The ill effect includes low performance in poultry production such as decreased weight gain, egg production, and hatchability. In addition to this inhibition of protein, DNA, and RNA synthesis, cytotoxicity, immunomodulation, cell lesions in the digestive tract, organs and skin, neural disturbances was also reported (Sokolovi M, et al. T-2 toxin incidence and toxicity in poultry. 2008. Arh Hig Rada Toksikol 59:43-52).
Patulin was reported to be isolated from fungus including Penicillium and Aspergillus. The effects of patulin were associated with alteration in renal function and inhibition of intestinal and renal ATPases (Puel O, Galtier P and Oswald I. P. 2010. Biosynthesis and Toxicological Effects of Patulin. Toxins. 2: 613-631).
The toxicity and clinical signs observed in animals when more than one mycotoxin is present in feed are complex and diverse. Mycotoxins are usually accompanied by other unknown metabolites which may have synergistic or additive effects. The ability of binders to alleviate the adverse effects of the several combinations of mycotoxins present naturally in feed on productivity and serum biochemical and hematological parameters remains yet to be explored.
Practical methods to detoxify mycotoxin contaminated grain on a large scale and in a cost-effective manner are not currently available. At present, one of the more promising and practical approaches is the use of adsorbents. However, several adsorbents have been shown to impair nutrient utilization (Kubena, L. F., Harvey R. B., Phillips T. D., Corrier D. E., and Huff W. E. 1990 Diminution of aflatoxicosis in growing chickens by the dietary addition of hydrated sodium calcium aluminosilicate. Poult. Sci. 69:727-735) and mineral adsorption (Chestnut, A. B., Anderson P. D., Cochran M. A., Fribourg H. A., and Twinn K. D. 1992. Effects of hydrated sodium calcium aluminosilicate on fescue toxicosis and mineral absorption. J. Anim. Sci. 70:2838-2846) and lack binding effects against multiple mycotoxins of practical importance (Edrington, T. S.; Sarr, A. B.; Kubena, L. F.; Harvey, R. B.; Phillips, T. D. 1996. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicology letter, 89: 115-122).
The use of mold inhibitors or preservation by acids can only reduce the amount of mold but does not influence the content of mycotoxins generated prior to treatment. If mycotoxins have been produced earlier they will not be affected in any form by mold inhibitors or acid mixtures, as they are very stable compounds. Thus these toxic compounds remain in the formerly infected commodity even if no further mold can be seen or detected. The most commonly used strategy of reducing exposure to mycotoxins is the decrease in their bioavailability by the inclusion of various mycotoxin binding agents or adsorbents, which leads to a reduction of mycotoxin uptake and distribution to the blood and target organs. Major advantages of adsorbents include expense, safety and the ease to add to animal feeds. Various substance groups have been tested and used for this purpose, with aluminum silicates, in particular clay and conventional zeolite minerals, as the most commonly applied groups.
Clay minerals have traditionally been supplemented to animal diets as multi-toxin binders. However, the degree of binding against high Log P value toxins such as ochratoxin A (OTA), mycophenolic acid and zearalenone was found to be low, possibly due to the charge and hydrophobicity of high Log P value mycotoxins. Hence the focus towards the use of alternate materials like zeolites (Dakovic, A., Tomasevic-Canovic, M., Dondur, V., Rottinghaus, G. E., Medakovic, V., and Zaric, S. (2005) Adsorption of mycotoxins by organozeolites. Colloids Surfaces B: Biointerfaces 46: 20-25), yeast cell wall products (Joannis-Cassan C, Tozlovanu M, Hadjeba-Medjdoub K, Ballet N, Pfohl-Leszkowicz A., (2011). Binding of zearalenone, aflatoxin B1, and ochratoxin A by yeast-based products: a method for quantification of adsorption performance. J Food Prot. 74:1175-85), molecular ion imprinting polymers (Yiannikouris A, Kwiatkowski A, Kudupoje M S and Matney C. Synthetic mycotoxin adsorbents and methods of making and utilizing the same. U.S. Pat. No. 8,426,541 B2) and functionalized material's (Dakovic, A., Tomasevic-Canovic, M., Dondur, V., Rottinghaus, G. E., Medakovic, V., and Zaric, S. (2005) Adsorption of mycotoxins by organozeolites. Colloids Surfaces B: Biointerfaces 46: 20-25) as toxin binders has been increased in recent past.
Zeolites which contain acidic sites at the surface with high surface area can bind organic molecules including toxins to a wide range of polarity. Both the H+ form and NH4+form of beta zeolite, which contain ordered and disordered frameworks, coexist and there are three mutually intersecting channels. The framework structure has two types of 12 membered ring pores. The channel system of zeolite beta has pore diameters of 5.6×5.6 Å and 7.7×6.6 Å (Barcia, P. S., Silva, J. A. C., Rodrigues, A. E., (2005) Adsorption Equilibrium and Kinetics of Branched Hexane Isomers in Pellets of Beta Zeolite. Microporous and Mesoporous Materials. 79: 145-163.). The present invention relates to the binding efficacy of H beta zeolite (HBZ) against mycotoxins, including pesticides.
It is understood that agricultural products, such as corn and soybean meal, are used as raw material source for the production of animal feed. During the cultivation and storage period, various classes of pesticides are added to these materials in order to protect them from the attack of pests. Belden, J. B., Gilliom, R. J., Martin, J. D. and Lydy, M. J., (2006) Relative Toxicity and Occurrence Patterns of Pesticide Mixtures in Streams Draining Agricultural Watersheds Dominated by Corn and Soybean Production. Integrated Environmental Assessment and Management. 3:90-100. Such usage of pesticides contaminates the entire food chain. Consumption of pesticide contaminated materials can affect animal performance and may lead to mortality. Sachin Kumar, Sharma, A. K., Rawat, S. S., Jain. D. K. and Ghosh, S. Use of pesticides in agriculture and livestock animals and its impact on environment of India. Asian Journal Of Environmental Science. 8(1):51-57; Fleischli, M. A., Franson, J. C., Thomas, N. J., Finley, D. L. and Riley, W. 2004. Avian Mortality Events in the United States Caused by Anticholinesterase Pesticides: A Retrospective Summary of National Wildlife Health Center Records from 1980 to 2000. Archives of Environmental Contamination and Toxicology. 46, 542-550.
In addition to this, pesticides tend to accumulate in the animal products meant for human consumption such as meat, egg, milk, etc., which is a serious health and regulatory concern across the world. Aulakh, R. S., Gill, J. P. S., Bedi, J. S., Sharma, J. K., Joia, B. S. and Ockerman, H. W. 2006. Organochlorine pesticide residues in poultry feed, chicken muscle and eggs at a poultry farm in Punjab, India. Journal of the Science of Food and Agriculture. 86:741-744; Nasreddine, L., Massin, D. P. 2002. Food contamination by metals and pesticides in the European Union. Should we worry?. Toxicology Letters. 127: 29-41. Based on a pesticides analysis from feed samples, the most frequently observed pesticides are carbofuran, thiram, and chlorpyrifos. The levels of these pesticides varied between 40 ppb and 100 ppb.
The widely used adsorbent to protect animals from the effect of pesticides is activated charcoal (AC). Frazier, D. I. 2000. Avian toxicology. In Manual of avian medicine, eds. G. H. Olsen and S. E. Orosz, 228-63. New York: Mosby. At a high level of contamination (above ppm levels) where mortality is an issue, AC is used irrespective of its nutrient binding ability. However, continuous usage of AC can lead to performance issues due to binding of nutrients. Review of mycotoxin-detoxifying agents used as feed additives: mode of action, efficacy and feed/food safety. CFP/EFSA/FEEDAP/2009/01. Thus, there remains a need for a pesticide binder that overcomes the drawbacks and performance issues associated with AC.